Role of the Active Site Cysteine of DpgA, a Bacterial Type III Polyketide Synthase

Claire C. Tseng, Shaun M. McLoughlin, Neil L. Kelleher, Christopher T. Walsh*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

DpgA is a bacterial type III polyketide synthase (PKS) that decarboxylates and condenses four malonyl-CoA molecules to produce 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) in the biosynthetic pathway to 3,5-dihydroxyphenylglycine, a key nonproteinogenic residue in the vancomycin family of antibiotics. DpgA has the conserved catalytic triad of Cys/His/Asn typical of type III PKS enzymes, and has been assumed to use Cys160 as the catalytic nucleophile to create a series of elongating acyl-S-enzyme intermediates prior to the C8 to C3 cyclization step. Incubation of purified DpgA with [14C]-malonyl-CoA followed by acid quench during turnover leads to accumulation of 10-15% of the DpgA molecules covalently acylated. Mutation of the active site Cys160 to Ala abrogated detectable covalent acylation, but the C160A mutant retained 50% of the V max for DPA-CoA formation, with a kcat still at 0.5 catalytic turnovers/min. For comparison, a C190A mutant retained wild-type activity, while the H296A mutant, in which the side chain of the presumed catalytic His is removed, had a 6-fold drop in kcat. During turnover, purified DpgA produced 1.2 equivalents of acetyl-CoA for each DPA-CoA, indicating 23% uncoupled decarboxylation competing with condensative C-C coupling. The C160A mutant showed an increased partition ratio for malonyl-CoA decarboxylation to acetyl-CoA vs condensation to DPA-CoA, reflecting more uncoupling in the mutant enzyme. The Cys-to-Ala mutant thus shows the unexpected result that, when the normal acyl-S-enzyme mechanism for this type III PKS elongation/cyclization catalyst is removed, it can still carry out the regioselective construction of the eight-carbon DPA-CoA skeleton with surprising efficiency.

Original languageEnglish (US)
Pages (from-to)970-980
Number of pages11
JournalBiochemistry
Volume43
Issue number4
DOIs
StatePublished - Feb 3 2004

ASJC Scopus subject areas

  • Biochemistry

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